PROJECT SUMMARY ? PROJECT 2 Cyclin D1 deregulation is observed frequently observed in human cancers especially in esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinomas (EAC). This deregulation implies that the cyclin D1/CDK4 kinase provides cells with a growth advantage, thereby contributing to cancer development. We have demonstrated that loss of Fbxo4, a component of the E3 ligase, SCFFBXO4, occurs in ESCC and FBXO4 knockout mice are susceptible to tumors. Additional work has revealed that FBXO4 regulates degradation of two distinct oncogenes in ESCC: cyclin D1 and FXR1. Overexpression of cyclin D1 increases mitogen- independent cell proliferation and FXR1 overexpression contributes to bypass of cell senescence and collective tumor initiation. In our new preliminary data, we have discovered that loss of FBXO4 results in cell addiction to Glutamine. In this context, glutamine appears essential due to its metabolic role, wherein it is metabolized to ?-ketoglutarate, an intermediate in the TCA cycle. Cells with mutant FBXO4 quickly undergo apoptosis under conditions of glutamine restriction. Given the generation of inhibitors of glutaminase, this observation provides a potential novel strategy for therapeutic intervention. These exciting discoveries provide critical insights into the mechanisms whereby FBXO4 suppresses neoplastic growth and support the notion that targets of FBXO4 such as FXR1 and cyclin D1/CDK4 activity are key esophageal cancer drivers. The identification of the SCFFBX4 as the cyclin D1 E3 ligase as well as our preliminary studies lead to the overarching hypothesis that the SCFFBX4 E3 ligase maintains threshold levels of the cyclin D1/CDK4 kinase and the FXR1, an RNA binding protein, to maintain esophageal tissue homeostasis. We hypothesize further that cyclin D1 and FXR1 overexpression foster ESCC progression, resulting in cellular vulnerabilities that can be rationally targeted. This hypothesis will be pursued through the following interrelated Specific Aims: (1) To determine the contribution of deregulated cyclin D1 to mutant FBXO4 driven glutamine-dependence in esophageal cancer; (2) To determine the mechanism(s) whereby mutant p53R172H cooperates with cyclin D1T286A to drive ESCC pathogenesis; and (3) To determine the therapeutic potential of targeting glutamine- metabolism in ESCC with dysregulated cyclin D/CDK4. Our collaborative work with Projects 1 and 2, along with support from the Core Facilities, provides new basic and translational perspectives on the cyclin D1/CDK4/CDK6 axis and glutamine metabolism in ESCC.